Tissue sample used for the cloning of ERCC1 gene was from the redundant tissue of surgical operation performed on a patient with brain injury. The patient gave written informed consent before enrolment. The present study was carried out following international and national regulation and approved by the Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University. PMD18-T vector used for TA cloning, PCDNA3.1 vector for cell transfection and pSilencer 4.1-CMV-neo vector for RNAi were obtained from Takara (Tokyo, Japan), Invitrogen (Carlsbad, CA, USA) and Ambion (Austin, TX, USA), respectively. TRIzol reagents, RevertAid™ First Strand cDNA Synthesis kit and Lipofectamine 2000 were from Invitrogen. Ex Taq DNA polymerase, restriction endonucleases BamH1 and EcoR1, and T4 DNA ligase were from Takara. Plasmid Mini kit was from Qiagen (Duesseldorf, Germany). M-per Mammalian Protein Extraction kit, Bradford protein assay kit and fluorescein isothiocyanate (FITC) reagents were purchased from Pierce (Rockford, IL, USA). Goat anti-human antibody ERCC1 and goat anti-human antibody β-actin used for western blot assays were from NeoMarkers (Fremont, CA, USA), and the secondary rabbit anti-goat antibody was from Fuzhou Maixin Biotech. Co., Ltd. (Fuzhou, China). Giemsa stain for colony-forming assays, propidium lodide (PI) fluorescence dye and RNase A used in the cell cycle assays were from Sigma (St. Louis, MO, USA) and Sango (Shanghai, China), respectively. All the reagents for high performance liquid chromatography (HPLC) assays were obtained from Merck (Darmstadt, Germany). Mouse anti-human monoclonal antibody (McAb) of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), as well as the corresponding secondary goat anti-mouse antibody used in the flow cytometry (FCM) were from Invitrogen.

Human NSCLC cell line H1299 was presented by Dr Zhe Zhang from Karolinska Institutet of Sweden. Human NSCLC cell line A549 was purchased from the cell bank of Peking Union Medical College (Beijing, China). NSCLC cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at the final concentration and maintained in a humidified atmosphere containing 5% CO₂ at 37°C. Cell culture reagents and FBS were obtained from Gibco (BRL, Long Island, NY, USA), antibiotics of penicillin and streptomycin were from Shandong Qilu Pharmaceutical Factory (Shandong, China), and Geneticin (G418) was from Sigma. Cisplatin and adriamycin (ADM) were purchased from Shandong Qilu Pharmaceutical Factory and Zhejiang Haimen Pharmaceutical Factory (Zhejiang, China), respectively.

ERCC1 gene was obtained from the tissue sample by reverse transcription-PCR (RT-PCR) technique. In brief, the tissue sample was incised and immediately put into liquid nitrogen. Total RNA was isolated from the samples using TRIzol reagent according to the manufacturer’s instructions. Approximately 0.5–5 μg of total RNA together with random hexamers were used for the cDNA synthesis through RT-PCR with RevertAid™ First Strand cDNA Synthesis kit. The final cDNA was used for the following PCRs. ERCC1 primers (forward, 5′-CGCGGATTCATGGA CCCTGGGAAGGACAAAG-3′; reverse, 5′-CCGGAATTCT CAGGGTACTTTCAAGAAGGG-3′) were designed with Primer 5 software and synthesized in Sangon Biotech Co., Ltd. PCR was performed in a 25-μl solution including 200 nM of the primers, 200 nM of converted cDNA, 10 mM deoxynucleotide triphosphates (dNTPs), 25 mM MgCl₂, 1.0 U Ex Taq DNA polymerase and the PCR buffer. The amplification thermal-cycling program consisted of 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec. The obtained PCR products were separated by the 1% agarose gel electrophoresis and the correct one was purified to be used in the following vector construction.

To obtain accurate and adequate target gene for vector construction, TA cloning was performed in a system with a total volume of 10 μl, including the purified PCR product 4 μl, PMD18-T vector 1 μl, salt solution 1 μl and ddH₂O 4 μl. After 5-min incubation at room temperature, it was transformed into the competent cell TOP 10. Positive stains were selected with blue-white selection, and cultured. Then, the plasmid DNA was extracted with the Plasmid Mini kit according to the manufacturer’s instructions. Target fragment in the plasmid DNA was isolated and inserted into the BamHI and EcoRI sites of PCDNA3.1 vector (5.5 kb) with the T4 DNA ligase. Thus, the connection product PCDNA3.1-ERCC1 plasmid was obtained, and then was transfected into the competent cell TOP 10 again. Positive stains of TOP 10 were selected by the blue-white selection and identified by PCR to exclude false clones, and further confirmed by sequencing identification. Then, the PCDNA3.1-ERCC1 plasmid obtained through the above vector construction processes was used in the following cell transfection experiments.

Parental H1299 cells 0.5 ml at the density of 0.6–1×10⁵/ml were seeded in 6-well plates per well and incubated at 37°C for 24 h until 80% confluent at the time of transfection. The transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions with slight changes. Plasmid PCDNA3.1-ERCC1 obtained above was transfected into H1299 cells; PCDNA3.1 vehicle and PEGFP-N1 plasmid with the green fluorescent protein (GFP) gene were transfected into the H1299 cells as parallel controls; and H1299 cells treated with transfection reagents without any plasmids or vehicles were used as the negative control. The transfection procedures were as follows: firstly, DNA-Lipofectamine complexes were prepared for each transfection sample. DNA [3.0 μl (1.0 μg)] and 5 μl Lipofectamine were mixed with 250 μl antibiotic- and FBS-free medium respectively. After 20 min at room temperature the mixtures were combined for 15–20 min. The final mixture was the DNA-Lipofectamine complexes. Then, cells in each well were washed with the antibiotic- and FBS-free medium and added with the above DNA-Lipofectamine complexes to incubate for 5 h at 37°C, then, 1 ml medium containing FBS were added into each well and incubated for further 24 h. The medium was then replaced by fresh normal medium and cells were cultured for 24–48 h to be used in the following screening and verification tests.

G418 was used to select the positive cells transfected with PCDNA3.1-ERCC1 and PCDNA3.1 vehicle, as the PCDNA3.1 vector contains the anti-G418 gene. The selecting concentration of G418 was determined as follows: briefly, H1299 cells (1×10³) grown in the antibiotics-free complete medium were seeded in 24-well plates and cultured till the cells attached. Then, they were treated with different concentrations of G418 (100–1,100 μg/ml with 100 μg/ml for one gradient) and continuously incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 10–14 days. The minimum G418 concentration at which all the cells were killed was used as the selection concentration in the transfection assays.

Total RNA was extracted with TRIzol reagent from the positive cells that had been screened by the G418 assays. cDNA was synthesized using the oligo(dt) primer according to the instructions of the RevertAid™ First Strand cDNA Synthesis kit. Subsequently, ERCC1 (441 bp) and PCDNA3.1 were amplified with the β-actin as an internal control. The primers were: ERCC1: 5′-GATGACCCAGATCATGTTTG-3′ (forward), 5′-TGGAGTTGAAGGTAGTTTCG-3′ (reverse); PCDNA3.1: 5′-CTGCTTACTGGCTTATC-3′ (forward) and 5′-GAAAGGACAGTGGGAGTG-3′ (reverse); β-actin: 5′-CAC CAACTGGGACGACAT-3′ (forward) and 5′-A CAGCCTGG ATAGCAACG-3′ (reverse). The amplification was performed in a 25-μl solution containing 200 nM of each of the primers, 200 nM the converted cDNA, 10 mM dNTPs, 25 mM MgCl₂, 1.0 U Ex Taq DNA polymerase and the PCR buffer, in conjunction with a thermal-cycle program consisting of 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec. Finally, the PCR products were separated on a 1% agarose gel and investigated in the Gel doc 2000 imaging and analysis system (Bio-Rad, Hercules, CA, USA).

Cells were washed with chilled PBS for three times and added to lysis buffer M-per mammalian protein extraction to incubate on ice for 30 min. The lysate was then centrifuged at 13,000 g for 5 min and the supernatant was collected. Concentration of total proteins was determined with the Bradford protein assay kit. Proteins were separated by SDS-PAGE and were transferred to a PVDF membrane. Primary antibodies goat anti-human antibody ERCC1 (1:200) and goat anti-human antibody β-actin (1:200) were applied to bond with target proteins on the PVDF membrane, and followed by the combination with the secondary antibody rabbit anti-goat (1:500). At last, the bands were identified by the enhanced FITC reagents.

Cells (1×10⁴) were seeded in 24-well plates with 18 duplications for each cell sample: H1299 cells (parental cells as control), H1299/E cells (transfected with PCDNA3.1-ERCC1) and H1299/V cells (transfected with PCDNA3.1 vehicle only). Survival cell of each cell sample were counted in three duplications every day. The procedures were as follows: briefly, culture solution in each well was removed with a straw. Cells left in the well were trypsinized with 100 μl 0.25% trypsin for 3–5 min, and then suspended uniformly with 800 μl RPMI-1640 medium and 100 μl trypan blue staining. Survival of the cells were evaluated under the inverted microscope (Olympus, Tokyo, Japan). Cell number was calculated according to the formula N=(n/4)x10⁴xV. Finally, cell growth curves were drawn with cell number as the y-axis and time as the x-axis.

Cells were uniformly seeded in 24-well plates at the density of 50, 100, 200 in triplicate, and cultured at 37°C in a humidified atmosphere containing 5% CO₂ for 5–6 days. The culture medium was replaced by fresh medium every 3 days. When colony contained >50 cells, it was washed with PBS, fixed with methanol for 15 min and stained with Giemsa for 30 min, then washed with water and dried in an airing chamber. Then, the colony was counted under the Gel doc 2000 imaging and analysis system. Colony forming rate was determined according to the equation: colony forming rate = (number of colonies/number of seeded cells) × 100%.

Cell cycle distribution was determined by flow cytometry (FCM). Cells were washed twice with PBS and fixed with 1 ml chilled 70% ethanol at 4°C overnight, then washed twice again with PBS and suspended at the density of 1×10⁶/ml with the staining solution containing 0.05 mg/ml PI and 0.1 mg/ml RNase A and incubated in the dark at 4°C for 30 min. At the end of the incubation period, the cell suspension was filtered with a 300 mesh screen to remove adhered cells. Finally, DNA content of each cell sample was detected on EPICS XL (Beckman, Urbana, IL, USA) and cell cycle distribution was determined with the muticycle software.

Matrigel invasion assays were performed to evaluate the ability of cell invasion in vitro. The lower surface of filter membrane (8 μm) of transwells was treated with 50 μl PBS supplemented with 5 μg fibronectin (Sigma) and dried at room temperature overnight. Matrigel (50 μl) that had been diluted to the density of 1.25 mg/ml with the cold serum-free RPMI-1640 medium was added to the upper surface of the filter membrane, incubating for 4–5 h. Cells, after three washes were trypsinized with 0.25% trypsin, and resuspended at the density of 1×10⁶/ml in the RPMI-1640 medium supplemented with 1% FBS. Cell suspension solution (100 μl) was added to the upper chamber of the transwells and 600 μl of the medium containing 1% FBS was added to the lower chamber, incubating at 37°C in a humidified atmosphere containing 5% CO₂ for 20–24 h. At the end of incubation period, cells in both chambers were treated with 10 and 60 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma), followed by 10 min dissolution with DMSO. Matrigel invasion ability of cells was determined through detecting the OD value of the dissolved solution at 450 nm.

Cisplatin response of cells was determined by MTT assays. Cells (1×10⁵/ml) were trypsinized with 0.25% EDTA and seeded into 96-well plates in triplicate. After incubated for 24 h, they were treated with different concentrations of cisplatin (0–400 μg/ml with 50 μg/ml for a gradient) for 24 h. At the end of the treatment period, 20 μl of MTT was added into each well and incubated for 4 h. The cells were then collected by the centrifugation and treated with 150 μl of DMSO for 10 min. The plates were scanned at 570 nm in a 96-well plate reader (DG3201, Nanjing, China). Each experiment was conducted three times in triplicate. Rate of inhibition (IR) were calculated by the formula IR (%) = (A_control − A_treatment)/A_control × 100%.

Drug accumulation in cells was assessed by detecting intercellular concentration of drugs with HPLC assays. After treated with 14 μg/ml cisplatin or 2 μg/ml adriamycin, cells (1×10⁶) were trypsinized and collected by centrifugation. Then cells were suspended with ddH₂O and lysed through the repeated freeze-thaw cycles. The lysate was centrifuged at 12,000 rpm for 5 min and the supernatant was collected to be analyzed with HPLC-10A VP (Shimadzu, Kyoto, Japan). The HPLC conditions for cisplatin analysis were as follows: chromatographic column: Shim-pack CLC-ODS (250×4.6 mm, 5 μm, Shimadzu, Kyoto, Japan); column temperature, 35°C; mobile phase, distilled water; test wavelength, 230 nm; flow-rate, 1.0 ml/min; and sample size, 10 μl. For ADM analysis, the HPLC conditions were chromatographic column: Shim-pack CLC-ODS (250×4.6 4.6 mm, 5 μm); column temperature, 40°C; mobile phase, the mixture containing methanol-0.01 M phosphate and 0.01% sodium dodecyl sulfate (v/v=85/15), adjusted to pH 2.5 with phosphoric acid; wavelength, 545 nm; flow-rate, 1.3 ml/min and sample size, 20 μl.

Expression of two membrane proteins, P-gp and MRP, were detected by the FCM. Cells were adjusted to the density of 5×10⁶–1×10⁷/ml with RPMI-1640 medium. Then, 100 μl of the cell suspension was mixed with 50 μl of the inactivated normal rabbit serum followed by the 10-min incubation at 4°C. At the end of incubation period, it was washed twice and centrifuged at 1,000 rpm for 5 min, and the supernatant was discarded. Then 5–50 μl of the chilled mouse antihuman McAb was added and incubated at 4°C for 30 min. After washed twice and centrifuged at 1,000 rpm for 5 min, the cells were suspended with 50 μl of the goat anti-mouse secondary antibody with fluorescent marker and incubated at 4°C for 30 min. After washing and centrifugation, the cells were resuspended with 0.5 ml of PBS, filtered with the 300 mesh screen and finally detected utilizing the FCM.

Human NSCLC cell line A549 with an intrinsic ERCC1 expression was selected as the parental cell in the RNAi experiments. Four small interfering RNAs (siRNAs) targeting to ERCC1 gene were designed according to the principles described previously (29). The sensestrand sequences of siRNA duplexes were as follows: siRNA897: 5′-CAGACCCTCCTGACCACATTTGG-3′; siRNA255: 5′-AAGCCCTTATTCCGATCTACACA-3′; siRNA783: 5′-AAGGCCTATGAGCAGAAACCAGC-3′; siRNA693: 5′-AAGGAGCTGGCTAAGATGTGAT-3′. In addition, one siRNA for the GAPDH gene was designed as a positive control: 5′-GUAUGACAACAGCCUCAAGTT-3′, and another siRNA not associated with ERCC1 gene but marked with flourescent indicator was the negative control: 5′-TTCTCCGAACGTGTCACGT-3′. They were synthesized and inserted into the BamH1 and HindIII sites of a pSilencer 4.1-CMV-neo vector, respectively, and followed by the transfection into the A549 cells utilizing Lipofectamine 2000. For ERCC1 gene, the gene-specific siRNA with optimal efficiency of interference was selected to be used in the following experiments. mRNA and protein expression of ERCC1 in the three cell samples, A549/E cells transfected with the siRNA of ERCC1, A549/V cells transfected only with the vehicle vector (as a positive control) and the parental A549 cells (as a negative control), was then detected by RT-qPCR and western blot assays, respectively. In addition, cisplatin response of A549/E cells and A549/V cells was also detected according to the above description in ‘Evaluation of cisplatin response’. The treatment concentration of cisplatin was 0–80 μg/ml with 10 μg/ml for a gradient.

The difference among three groups was calculated by one-way ANOVA, and the difference between two groups was analyzed by Student’s t-test. All of the analysis was performed on SPSS 19.0 software. A value of P<0.05 was considered as statistically significant.

To investigate the biological functions of ERCC1 on NSCLC cells, ERCC1 gene was cloned and transfected into the NSCLC H1299 cells with no intrinsic ERCC1 expression. ERCC1 gene was attained by RT-PCR technique (Fig. 1A) and linked to the PCDNA3.1 vector, to compose the recombinant plasmid PCDNA3.1-ERCC1 which was transfected into the competent cell TOP 10 treated with chilled CaCl₂ solution. Positive TOP 10 clones were screened by blue-white selection and further subject to PCR identification with the primers of ERCC1 gene. As a result, a band of 894 bp coincided with the target gene ERCC1 (Fig. 1B) was successfully amplified in clone 2 which was then selected to the following confirmation assays in which PCR identification was conducted again with the primers of PCDNA3.1. Two bands 894 bp (ERCC1) and 5.5 kb (PCDNA3.1) were indicated in the result of the restriction enzyme cleave identification (Fig. 1C), which demonstrated that the recombinant plasmid of PCDNA3.1-ERCC1 had been successfully cloned.

G418 was used as the screening regent in the following eukaryotic transfection experiments. The screening concentration was determined as 600 μg/ml by the assays of cytotoxic reaction on H1299 cells (Fig. 1D). The recombinant plasmid PCDNA3.1-ERCC1 and PCDNA3.1 vehicle were, respectively, transfected into the H1299 cells. They were named H1299/E and H1299/V, respectively. H1299 cells transfected with the GFP gene pEGFP-N1 (H1299/GFP cells) and the parental H1299 cells without DNA transfection were the positive and negative controls, respectively. The expression of GFP detected in the H1299/GFP cells (Fig. 1E) indicated that the processes of transfection had been accurately carried out. Following G418 screening with the concentration of 600 μg/ml, the living clones (Fig. 1F) were selected and cultured to an extended scale.

Further identification for the transfection was conducted with PCR technique. ERCC1 expression (441 bp) appeared in H1299/E cells not in H1299/V and H1299 cells when the primers of ERCC1 were used in the PCR process (Fig. 1G), by contrast, when the primers of PCDNA3.1 were applied, the expression of ERCC1-PCDNA3.1 (1,269 bp) and PCDNA3.1 (364 bp) were detected in H1299/E and H1299/V cells, respectively, and no expression of either was found in H1299 cells (Fig. 1H). Correspondingly, the protein expression of ERCC1 was also demonstrated in H1299/E cells, not in H1299/V and H1299 cells by the western blot analysis (Fig. 1I). These findings indicated that ERCC1 gene had been successfully transfected into H1299 cells.

Cell proliferation of H1299/E, H1299/V and H1299 cells was evaluated by survival cell count assays. H1299/E and H1299/V cells displayed a similar tendency in the growth curve, they showed no significant difference with their parental cells H1299 in growth rate with P>0.05, although they had a slightly slower growth compared with the parental cells (Fig. 2A). Therefore, cell proliferation of NSCLC H1299 cells in the present study was not affected by the expression of ERCC1.

Colony-forming assays were also employed to examine the ability of cell proliferation as well as the population dependence of H1299/E and H1299/V cells. Consequently, the two cell lines exhibited no significant difference in the colony-forming rate with 25.1±4.6 and 27.6±5.4%, respectively and P>0.05 (Fig. 2B). The finding demonstrated that ERCC1 expression had no effect on the deterioration of the H1299 cells.

Cell cycle distribution in each cell lines of H1299/E, H1299/V and 1299 was examined by FCM technique. The results suggested that there was no significant difference in cell cycle distribution among the three cell lines (Fig. 2C), indicating that the expression of ERCC1 had no effect on the cell cycle of the H1299 cells.

Cell invasion is a common biological behavior of cancer cells and was investigated by Matrigel invasion assays in the present study. The invasive ability of the cells was determined by the ratio of the cells in the upper and lower surface of the transwell. Then, the relatively invasive ability of H1299/E and H1299/V cells compared with their parental H1299 cell was calculated, respectively. There was no significant difference in the invasive ability between H1299/E and H1299/V cells (Fig. 2D). The finding indicated that the expression of ERCC1 did not affect the invasive ability of H1299 cells in vitro.

MTT assays were performed to detect the response of H1299/E and H1299/V cells to cisplatin, respectively. The IC₅₀ of cisplatin in the H1299/E cells was 186 μg/ml, 4.23 times higher than that of H1299/V cells which was only 44 μg/ml (Fig. 3A). The results suggested that H1299 cells developed an evident cisplatin resistance after transfection with ERCC1 gene.

To clarify whether the acquired cisplatin resistance of H1299/E cells was correlated with drug influx or efflux, intercellular accumulation of cisplatin in H1299/E and H1299/V cells was detected by means of HPLC. Firstly, the standard curve for cisplatin was established with the correlation coefficient of 0.9999, suggesting that the detection system of HPLC with a high confidence was available. Then, intercellular cisplatin concentration in H1299/E and H1299/V cells was tested and calculated based on the established standard curve. They were 3.41 and 4.24 μg/ml, respectively, with no significant difference between the two cell lines (P>0.05, Fig. 3B). By contrast, intercellular concentration of ADM in the two cell lines was determined as well, and the same result was obtained, which indicated that the results produced by HPLC for cisplatin was credible. Consequently, the resistance of H1299/E cells to cisplatin in the present study may be not associated with drug influx or efflux.

As further confirmation of the above, we detected the expression of the associated membrane proteins, P-gp and MRP, which have been reported to participate in drug resistance (12). The FCM result showed that there was no significant difference in the expression of P-gp and MRP proteins between H1299/E and H1299/V cells. Therefore, cisplatin resistance of H1299/E cells in the present study may be not associated with the expression of P-gp and MRP proteins.

To further confirm the effect of ERCC1 expression on the cisplatin response of NSCLC cells, RNAi technique was employed to silence the ERCC1 expression of A549 cells. Four siRNAs were designed, synthesized, and transfected into A549 cells to interfere with the ERCC1 expression, siRNA255 was selected for use in the following experiments (Fig. 4A). As the parallel positive control, GAPDH was silenced in A549/G cells transfected by the siRNA to GAPDH, and normally expressed in A549/E-255 and parental A549 cells (Fig. 4B). The results demonstrated the effectiveness of the siRNA transfection processes utilized in the present study.

Following the transfection, RT-PCR and western blot assays were conducted to detect the mRNA and protein expression of ERCC1, respectively, in A549, A549/V and A549/E cells. Compared with parental A549 and A549/V cells, mRNA (Fig. 4C) and protein (Fig. 4D) expression of ERCC1 in A549/E cells was significantly suppressed, suggesting that NSCLC cell model with the silenced ERCC1 (A549/E) was successfully established and could be used in the following cytotoxicity assays.

Cell proliferation of A549/E and A549/V cells treated with cisplatin was detected by MTT assays. The IC₅₀ of cisplatin in A549/E cells was 18.2 μg/ml, much less than that in A549/V cells at 53.4 μg/ml. The result indicated that the suppression of ERCC1 expression by siRNA significantly enhanced the sensitivity of A549 cells to cisplatin, thus demonstrating that ERCC1 is implicated in the processes of cisplatin resistance of NSCLC cells.